The Ca2+ ion was first proposed as an intracellular messenger by Rasmussen in 1970 (Science 170:404–12), and now is known to be responsible for regulation of amazingly diverse physiological processes ranging from control of blood pressure and muscle contraction, release of insulin and other hormones, to immune and neurological memory (Bootman et al, Semin. Cell Dev. Biol. 12:3–10(2001); Clapham and Sneyd, Adv. Second Messenger Phosphoprotein Res. 30:1–24 (1995)). Moreover, elevation of Ca2+ frequently occurs in the same cell in response to different signals with distinct consequences depending on the nature of the stimulus.
A general explanation for “broadband” signaling through Ca2+ is that the molecules involved in Ca2+ homeostasis are segregated into spatially privileged compartments minimizing uncontrolled crosstalk between signaling pathways (Berridge et al, Science 287:1604–1605 (2000)). Intracellular Ca2+-release is primarily mediated by two families of Ca2+-release channels, ryanodine (RyR) and inositol 1,4,5-trisphosphate receptors (IP3R), which are localized to the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane. Ca2+ released from ER stores through these channels diffuses submicron distances before being rapidly buffered or sequestered into mitochondria and/or the ER/SR lumen via the SR/ER CaATPase (SERCA) (Allbritton et al, Science 258:1812–1815 (1992)). These cycles of elementary Ca2+ flux are the basis for local signaling events, and also represent the basic unit for global Ca2+ events such as cardiac muscle contraction and relaxation (Ju and Allen, J. Physiol. 516:793–804 (1999)). Elevations in intracellular IP3 increase the frequency of elementary release of Ca2+ from the ER/SR (‘blips’).
Based on the Ca2+-induced calcium-release (CICR) properties of IP3R and RyR, localized elevation of Ca2+, if concentrated near adjacent Ca2+-release channels, can trigger additional Ca2+-release leading to a considerably larger local release of Ca2+ (‘puffs’). On their own, these puffs are instrumental in local Ca2+ regulatory mechanisms including exocytosis and ion channel activation (Rottingen and Iversen, Acta Physiol. Scand. 169:203–219 (2000)). However, the concerted action of many Ca2+ puffs can activate local RyRs leading to Ca2+ waves spreading across large distances. As a result, the generation of Ca2+ waves and intracellular Ca2+ signaling is dependent on the coupling of highly localized Ca2+-release events to global Ca2+ signaling mechanisms.
Regulation of specific Ca2+-dependent cell signaling events is mediated by the tight spatial control of microdomain Ca2+ concentrations due to the organization of Ca2+-release proteins within the ER/SR in relation to each other, Ca2+-effector proteins (ion channels, kinases), Ca2+-uptake mechanisms (SERCA, mitochondria), as well as Ca2+-buffering proteins. Therefore, the proper function of Ca2+-release channels and thus, calcium, in a physiological context requires the appropriate spatial segregation of these proteins in cells.
Ankyrins are a family of membrane-associated proteins recently demonstrated to be required for targeting of ion channels to physiological sites in specialized membrane domains (Bennett and Chen, Current Opinion in Cell Biology 13:61–67 (2001)). Indeed, a role for ankyrin-B in cellular targeting of Ca2+-release channels has recently been proposed based on observations that ankyrin biochemically interacts with RyR and IP3R (Bourguignon et al, J. Biol. Chem. 270:7257–7260 (1995); Bourguignon et al., J. Biol. Chem. 268:7290–7297 (1993); Hayashi and Su, Proc. Natl. Acad. Sci. USA 9:9 (2001); Joseph and Samanta, J. Biol. Chem. 268:6477–6486 (1993)), and that ankyrin-B null mice exhibit abnormal localization of calcium homeostasis proteins in cultured neonatal cardiomyocytes (Tuvia et al, J. Cell Biol. 147:995–1008 (1999)).
The present invention results, at least in part, from studies demonstrating that ankyrin-B is necessary for the subcellular segregation and/or trafficking of IP3R to specialized cellular sites, ultimately regulating spatially-privileged Ca2+ dynamics via a conserved mechanism utilized in multiple cell types.